Engineering and Characterization of a Long-Half-Life Relaxin Receptor RXFP1 Agonist

Relaxin-2 is a peptide hormone with important roles in human cardiovascular and reproductive biology. Its ability to activate cellular responses such as vasodilation, angiogenesis, and anti-inflammatory and antifibrotic effects has led to significant interest in using relaxin-2 as a therapeutic for heart failure and several fibrotic conditions. However, recombinant relaxin-2 has a very short serum half-life, limiting its clinical applications. Here, we present protein engineering efforts targeting the relaxin-2 hormone in order to increase its serum half-life while maintaining its ability to activate the G protein-coupled receptor RXFP1. To achieve this, we optimized a fusion between relaxin-2 and an antibody Fc fragment, generating a version of the hormone with a circulating half-life of around 3 to 5 days in mice while retaining potent agonist activity at the RXFP1 receptor both in vitro and in vivo.


Figure S3 :
Figure S3: Sites of relaxin-2 engineering in Fc-relaxin-2 fusion constructs.a,c, Docking model of relaxin-2 bound to the leucine-rich repeats (LRRs) of RXFP1's ectodomain 1 .b, Details of the relaxin-2-LRR interface.In magenta are RXFP1 residues involved in the binding interface, in green are the "relaxin-binding cassette" residues of relaxin-2's B-chain, Arg13, Arg17, and Ile20, and in blue are the Met residues mutated to Lys in SE301.The Trp28 residue was not included in the docking model.The model shows that the Met residues on the relaxin-2 B-chain are not positioned near the binding interface.d, Based on the model, the position of the single-chain relaxin-2 "mini-C" linker likely does not interfere with the binding of relaxin-2 to RXFP1.

Figure S4 :
Figure S4: SE301 signaling activity at human RXFP2 and mouse RXFP1.a, CRE-SEAP G s signaling assay data for SE301 compared to native relaxin-2 at human RXFP2.Data are normalized to the native relaxin-2 response at human RXFP1 and are mean ± s.e.m. from technical triplicates.b, CRE-SEAP G s signaling assay data for SE301 compared to native human relaxin-2 at mouse RXFP1.Data are normalized to the native human relaxin-2 response at mouse RXFP1 and are mean ± s.e.m. from technical triplicates.

Figure S5 :
Figure S5: SE301 versus native relaxin-2 activity in the G s GloSensor cAMP assay.a-b, GloSensor G s signaling assay data for native relaxin-2 (a) and SE301 (b) with measurements taken before ligand addition, and 5, 10, 15, 20, 25, and 30 minutes after ligand addition.Data are normalized to the native relaxin-2 response at human RXFP1 and are mean ± s.e.m. from technical triplicates.

Table S1 : CRE-SEAP G s signaling assay EC 50 and E max data for Figure 1c
† Mean ± s.e.m., n=3 technical replicates.

Table S2 : CRE-SEAP G s signaling assay EC 50 and E max data for Figure S1c
† Mean ± s.e.m., n=3 technical replicates.

Table S3 : CRE-SEAP G s signaling assay EC 50 and E max data for Figure S2
† Mean ± s.e.m., n=3 technical replicates.

Table S4 : CRE-SEAP G s signaling assay EC 50 and E max data for Figure S4
† Mean ± s.e.m., n=3 technical replicates.

Table S5 : GloSensor G s signaling assay EC 50 and E max data for Figure S5
† Mean ± s.e.m., n=3 technical replicates.